Simultaneous Co-administration Research Articles

The Co-Delivery of Oxaliplatin Abrogates the Immunogenic Response to PEGylated siRNA-Lipoplex

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex. Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated. Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses. Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.

Herb–drug interaction of Fucus vesiculosus extract and amiodarone in rats: A potential risk for reduced bioavailability of amiodarone in clinical practice

Fucus vesiculosus is a seaweed claimed to be useful for obesity management. Therefore, considering the relationship between obesity and cardiovascular diseases, this work aimed to assess the potential for an herb–drug interaction among a standardized F. vesiculosus extract (GMP certificate) and amiodarone (a narrow therapeutic index drug) in rats. In a first pharmacokinetic study, rats were simultaneously co-administered with a single-dose of F. vesiculosus (575mg/kg, p.o.) and amiodarone (50mg/kg, p.o.); in a second study, rats were pre-treated during 14days with F. vesiculosus (575mg/kg/day, p.o.) and received amiodarone (50mg/kg, p.o.) on the 15th day. Rats of the control groups received the corresponding volume of vehicle. After analysis of the pharmacokinetic data it deserves to be highlighted the significant decrease in the peak plasma concentration of amiodarone (55.4%) as well as the reduction of systemic exposure to the parent drug (∼30%) following the simultaneous co-administration of F. vesiculosus extract and amiodarone. This paper reports, for the first time, the herb–drug interaction between F. vesiculosus and amiodarone, which determined a considerable decrease on amiodarone bioavailability in rats. Therefore, the therapeutic efficacy of amiodarone may be compromised by the concurrent administration of herbal slimming medicines/dietary supplements containing F. vesiculosus.

Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis-using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling-revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.

Interaction between the cardiovascular effects of Clonidine and the κ-opioid agonist U-50,488H in the anterior hypothalamic area of the rat brain

In thiobutabarbitone-anaesthetized rats, microinjection of clonidine (1-40 nmol) into the anterior hypothalamic area (AHy) produced dose-dependent reductions in mean arterial blood pressure and heart rate. Microinjection of the kappa-opioid agonist U-50,488H (3 and 10 nmol) did not modify these parameters. Simultaneous co-administration of clonidine (4 nmol) and U-50,488H (10 nmol) into the AHy resulted in significant potentiation of the clonidine-induced hypotension and marked attenuation of the bradycardia. A lower dose of U-50,488H (3 nmol) co-administered with clonidine (4 nmol) did not influence the cardiovascular responses to clonidine. These findings suggest that AHy neurons involved in the cardiovascular responses to clonidine may be modulated by kappa-opioid receptor stimulation.

The Effect of the Parenteral Route of Administration on the Immune Response to Simultaneous Nasal and Parenteral Immunizations Using a New HBV Therapeutic Vaccine Candidate

Chronic hepatitis B is a major health problem, with more than 350 million people infected worldwide. Available therapies have limited efficacy and require long-term continuous and expensive treatments, which often lead to the selection of resistant viral variants and rarely eliminate the virus. Immunotherapies have been investigated as a promising new approach. Several vaccine formulations have been clinically tested in chronic patients, none of which have clearly demonstrated efficacy so far. In this study we evaluated a new vaccination strategy comprising the simultaneous co-administration by the nasal and parenteral routes of a multicomponent vaccine formulation in BALB/C and HBsAg-transgenic mice. The formulation under study contains the surface and nucleocapsid antigens of the HBV, and was co-administered by the nasal route and three parenteral routes. For parenteral administration we also evaluated the immunogenicity of the antigenic mixture with alum or without the adjuvant. The immune response was evaluated by ELISA and IFN-γ ELISPOT assays. Our results indicate that all variants generated a strong antibody response in the sera against both antigens, but differed in their capacity to induce cellular immune responses against the surface antigen. Mice immunized by the nasal and subcutaneous routes without alum generated the highest IFN-γ-secreting CD8+ T-cell response, and results in this transgenic mouse model showed that there is no need to include alum. In conclusion, our results indicate that the immunization routes have to be carefully selected before carrying out clinical trials to optimize the immune response and promote further clinical development.

607 Exploring chemoresistance in vivo; are we making progress

Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis—using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling—revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.

Essential phospholipids protection against mercury uptake and histopathological changes in the intestine of fish, Oreochromis mossambicus (Trewavas)

Histochemical examination of fingerlings and adults of freshwater teleost Oreochromis mossambicus (Trewavas) exposed to sublethal concentration of HgCl2 (0.15 mg/l) for 30 days revealed considerable uptake of mercury by their respective intestines. Simultaneous co-administration of Essential phospholipids (EPL) along with food to the fishes, significantly supressed mercury uptake by the intestinal tissues, except the goblet cells which were still loaded with Hg in both stages. Due to accumulated mercury visible histopathological damage was seen in muscle layers, lamina propria and basement membrane of columnar cells. No noticeable damage in intestine was seen, when EPL was simultaneously fed to fishes along with HgCl2 exposure. Results suggest that EPL plays prophylactic role against metal uptake and structural damage in fish intestine exposed to inorganic mercury.

Acute Migraine Treatment With Oral Triptans and NSAIDs in a Managed Care Population

To describe the use of oral triptans with or without nonsteroidal anti-inflammatory drugs (NSAIDs) for acute migraine treatment in a managed care population and its potential impact on functionality. Prescription or over-the-counter NSAIDs with or without oral triptans are commonly used for treatment of acute migraine pain. Little is known about patients' treatment strategy when they have had experiences using NSAIDs and oral triptan cotherapy and the relationship between treatment strategy and migraine symptoms and functionality. Migraineurs identified from an administrative claims database were surveyed for their use of oral triptans and NSAIDs during their last attack in the screening phase and during the subsequent migraine attack in the follow-up phase of the study. Treatment regimens were classified into 6 categories: simultaneous coadministration of triptans and NSAIDs (T and N); triptans first followed by NSAIDs (T N); NSAIDs first followed by triptans (N T); triptans only (TRP only); NSAIDs only (NSAID only); and others. Headache experience, reasons for treatment regimens, and treatment satisfaction were cross-tabulated by treatment regimens. The log-rank test was used for the analysis of time-to-event data. Among 8440 oral triptan users surveyed during the screening phase, 2307 (27%) reported using triptans and NSAIDs combination therapy during their last migraine attack. Of those, 1502 experienced a subsequent migraine attack and completed the follow-up survey; 38% of these 1502 patients who used triptans and NSAIDs cotherapy during their last migraine attack continued to use combination therapy for their next attack. The most common treatment regimen, excluding "others" (n = 354, 24%), was TRP only (n = 403, 27%), followed by N T (n = 345, 23%), NSAID only (n = 170, 11%), T&N (n = 152, 10%), and T N (n = 75, 5.0%). More TRP only patients became nausea-free within 1 h after an initial dosing. TRP only, T&N, and N T had significantly shorter median hours of suffering from migraine and limited functioning, as compared with other treatment regimens. Substantially more patients taking TRP only (34.7%) were very satisfied with their current treatment regimen than other regimens. Migraine patients frequently change their treatment regimens in response to headache profiles. For patients with migraine associated nausea symptom, combination of therapy with triptan and NSAIDs appears to be less effective in relieving nausea than triptan monotherapy. Triptan montherapy remains a common and an effective migraine treatment strategy.

Characterization of Pain, Vasculature and Innervation in Sickle Cell Disease.

Pain in sickle cell disease (SCD) is characterized by chronic vasculopathy. Characterization of pain and vasculature may be critical to improve the analgesic ability of opioids in treating pain in SCD. Therefore, we examined the association between vasculature, innervation and pain in a transgenic mouse model of SCD (BERK) and control mice (HbABERK). Pain behavior was analyzed using paw withdrawal latencies (PWL) using Hargreave's device for thermal hyperalgesia. A radiant heat stimulus was applied to the plantar surface of each hind paw from underneath the glass floor with a projector lamp bulb (CXL/CXR, 8 V, 50 W). PWL to the nearest 0.1 s was automatically recorded upon paw withdrawal. Paws were alternated randomly to preclude “order” effects. We observed that 5 mo old sickle mice showed significantly lower thresholds to noxious heat than controls (p=0.002 for females; and p=0.04 for males; n=5–7 and 15 observations/mouse). Females showed a significantly shorter latency than their respective males (control, p=0.03 and sickle, p=0.01), i.e., they were more sensitive to thermal stimuli. No significant difference was observed between sickle and control nor male and female mice (p > 0.05, n = 5–7) for mechanical allodynia using von Frey filaments, suggesting that sickle mice show increased sensitivity to thermal hyperalgesia. BERK mice were subcutaneously injected with 0.7 mg morphine/Kg mice/day (equivalent to 50 mg/70 kg /day/ human adult) with an added increment of 0.14 mg/day/Kg for each week for three different time periods; Group I, treated for 6 weeks, Group II treated for 6 weeks followed by withdrawal for 6 weeks and Group III treated for 12 weeks. Morphine treatment for 6 weeks as well as 12 weeks resulted in a ∼50–75% increase in PWL vs PBS, suggesting that chronic morphine treatment decreased hyperalgesia in sickle mice. The anti-hyperalgesic effect of morphine was antagonized by simultaneous co-administration of naloxone (2 mcg/Kg/day), suggesting an opioid receptor mediated effect. Naloxone treatment alone did not show any significant effect on PWL. In Group II, withdrawal of morphine for 6 weeks following 6 wks of treatment showed about 35% increase in PWL vs PBS (p<0.005), suggesting that the anti-hyperalgesic effect of morphine continues after its withdrawal. The continued anti-hyperalgesic effect of morphine following withdrawal could be secondary to its vasoregulatory effect. Indeed, confocal microscopy of skin sections revealed disorganized and stringy blood vessels, nerves and lymphatics in sickle mice vs. control. Deep blood vessels and nerve plexus, as well as the skin, were appreciably thicker in controls vs sickle. The diameter of lymphatics and densities of both blood vessels and nerves were significantly lower in sickle vs HbA controls (p= 0.0001 and 0.002, for sub-epidermal and dermal blood vessels, respectively; p=0.04 for lymphatic diameter; p=0.0001 for sub-epidermal, dermal and deep dermal nerve fibers). Functionally, we observed about 30% decrease in subcutaneous O2 measured by Laser Doppler (in real-time) in BERK vs HbA control (p<0.05). Thus, complementary alteration in vasculature and innervation may underlie the chronic pain in SCD. Given that morphine promotes angiogenesis and vasodilation, its prolonged anti-hyperalgesic activity could be due to its vasoregulatory function. Together, these data suggest that intermittent therapy with morphine in SCD may provide analgesia similar to continued therapy and may even be devoid of side-effects.

Multidomain Synthetic Peptide B2A2 Synergistically Enhances BMP-2 In Vitro

A multidomain, synthetic peptide designated B2A2 synergizes the activity of BMP-2. B2A2 interacts with BMP receptor isoforms, potentiating the action of BMP-2 in activating alkaline phosphatase and triggering Smad and MAPK signaling. B2A2's design permits its delivery as a local surface coating as well as a soluble co-factor, thus broadening potential bioengineering applications. BMP-2 induces osteogenic differentiation and accelerates bone repair. Although BMP-2 inhibitors have been discovered, no BMP-2 mimetics or enhancers that function in the physiological range have yet been found. Here we report that a synthetic peptide designated B2A2, consisting of (1) a BMP receptor-targeting sequence, (2) a hydrophobic spacer, and (3) a heparin-binding sequence, is a positive modulator of recombinant BMP-2. Cultures of mesenchymal cell lines C2C12 and C3H10T1/2 were given B2A2, recombinant BMP-2, or both. Alkaline phosphatase (ALP) activity was assayed by conversion of paranitrophenol phosphate (PNPP). Signaling through Smad and MAP kinase pathways was monitored by Western blot. Receptor binding was assessed by incubating immobilized B2A2 with soluble recombinant receptor-Fc chimeras and detecting bound receptor by anti-Fc antibody ELISA. Surface coating of medical device materials was done by first dip-coating with silyl-heparin, followed by B2A2. Treatment of cells with B2A2 alone marginally increased ALP activity. However, B2A2 plus BMP-2 resulted in 5- to 40-fold augmentation of ALP compared with BMP-2 alone in C3H10T1/2 or C2C12 cells, respectively. This synergistic enhancement was observed over a broad concentration range (4-1000 ng/ml BMP-2). B2A2 interacted directly with BMP receptor isoforms (preferentially to BMPR-Ib and ActivinR-II). In cells, B2A2 + BMP-2 led to a repression of MAP kinase and an increase of Smad activation, consistent with known activation pathways of BMP-2. B2A2 was ineffective when paired with other cytokine/growth factors (basic fibroblast growth factor [FGF-2], TGF-beta1, vascular endothelial growth factor [VEGF]). Simultaneous co-administration was not strictly required. Pulse-chase experiments revealed that temporal separations up to 1 h were still effective. B2A2 was also effective when delivered in a polystyrene- or stainless steel-coated surface through a heparin platform (silyl-heparin) while BMP-2 was added exogenously in solution. These results suggest that B2A2 might promote aggregation of receptor subunits, enabling BMP-2 to activate signaling pathways at effectively lower concentrations. Synthetic multidomain constructs like B2A2 may be useful to accelerate bone repair/deposition through augmentation of endogenous levels of BMP-2 or through local BMP-2 contained in artificial or engineered matrices.

Antifeedant bioactivity of sesquiterpene lactones from neurolaena lobata and their antagonism by γ-aminobutyric acid

The germacranolides and furanoheliangolides isolated from aerial parts of Neurolaena lobata (L.) R.Br. are assessed as antifeedants against fifth instar larvae of Spodoptera litura (Noctuidae) using leaf disc bioassays. In terms of potency, they are less active than parthenolide, more active than picrotoxinin, and comparable to strychnine. The antifeedant effect of neurolenin B, appears to result from interaction with GABA A receptors in the chemosensilla of S. litura as suggested for the known GABA antagonists strychnine and picrotoxinin. All three compounds show strong antifeedant activities, which are completely lost after simultaneous co-administration of equimolar GABA.

Simultaneous analysis of diphenylmethoxyacetic acid, a metabolite of diphenhydramine, and its deuterium-labeled stable isotope analog in ovine plasma and urine.

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([2H10]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography-electron-impact mass spectrometry (GC-EI-MS) analysis of DPMA and [2H10]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-microliter aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [2H10]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [2H10]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [2H10]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [2H10]DPMA in biological samples obtained from sheep (e.g. plasma and urine).

Effects of continuous stem cell factor administration on normal and erythropoietin‐stimulated murine hemopoiesis: Experimental results and model analysis

The aim of this study was to determine how stem cell factor (SCF) modifies hemopoietic cell production. First we determined the effects of a prolonged SCF administration on murine hemopoiesis and analyzed the results by a mathematical simulation model of hemopoiesis in order to explain the data. Subsequently we investigated the effects of simultaneous coadministration of SCF+erythropoietin (Epo), to see how effects of early and late cytokines superimpose. SCF administration during 14 days induced a proliferative wave through the hemopoietic system; colony forming units-granulocyte macrophage (CFU-GM), burst forming units-erythroid (BFU-E) and colony forming units erythroid (CFU-E) were the first to be augmented, followed by their respective progeny, ultimately leading to increased blood cell numbers. Despite continued treatment most cell numbers returned to normal values in 14 days, colony forming units-spleen (CFU-S), however, remained elevated. This wave pattern could be explained within the framework of a previously established mathematical model of hemopoiesis, if it was assumed that SCF affected the cycling status of primitive cells and if regulatory feedback loops of erythroid and myeloid progenitors on these cells were also allowed. Simultaneous SCF and Epo administration led to synergistic effects on CFU-E numbers and hematocrit values at moderate Epo doses. At high Epo doses, however, this was less pronounced. We conclude that SCF increases the input into committed hemopoietic lineages, where late acting cytokines can induce further amplification.

Differential actions of central alloxan upon opioid and nonopioid antinociception in rats: A further examination

Previous work demonstrated that central pretreatment with alloxan significantly reduced antinociception induced by morphine and 2-deoxy-D-glucose (2DG), an opioid-mediated stressor, but not induced by continuous cold-water swims (CCWS), a nonopioid-mediated stressor. The alloxan-induced deficits in 2DG antinociception were ameliorated by coadministration of D-glucose (3 M, 3M-DG). The present study evaluated this relationship further by: 1. a) examining whether central alloxan reduced morphine antinociception following either simultaneous 3M-DG and alloxan coadministration, alloxan followed 10 days later by 3M-DG and 3M-DG alone, and 2. b) determining whether central alloxan pretreatment altered nonopioid antinociception induced by the muscarinic cholinergic agonist, pilocarpine. Morphine (2.5–5 mg/kg, SC) antinociception on the tail-flick and jump tests was significantly reduced by central alloxan. In contrast, simultaneous coadministration of 3M-DG and alloxan failed to alter morphine antinociception. This ameliorative effect of 3M-DG was not due to its ability to affect morphine antinociception, and was time-dependent in that delays in 3M-DG administration failed to affect the alloxan-induced deficit. Central alloxan pretreatment failed to alter pilocarpine antinociception on the tail-flick test, and increased pilocarpine antinociception on the jump test. That central alloxan reduced opioid (e.g., morphine and 2DG), but not nonopioid (e.g., CCWS, pilocarpine) forms of antinociception suggests a specific mode of action, possibly through disruptions of glucoprivic control mechanisms which is in keeping with the suggestion that opioid systems are sensitive to changes in central glucose function.

Effect of ascorbic acid or thiamine on acetaldehyde, disulfiram-ethanol- or disulfiram-acetaldehyde-induced mortality.

Ascorbic acid or thiamine was administered, i.p., to male mice 1 1/2 hours prior to i.p. administration of acetaldehyde. Ascorbic acid reduced acetaldehyde mortality at doses between 16 mmole/kg to 18 mmole/kg. Thiamine reduced acetaldehyde-induced mortality at lower acetaldehyde doses (16 mmole/kg) but not at higher doses (17 and 18 mmole/kg) that induced 100% mortality. Simultaneous coadministration of acetaldehyde and ascorbic acid caused a reduction in mortality. The effect of ascorbic acid or thiamine pretreatment on disulfiram-ethanol-induced mortality was investigated. A single dose of thiamine (0.24 mmole/kg) given 1 1/2 hours prior to administration of ethanol (11 mmole/kg to 88 mmole/kg) in disulfiram-treated mice reduced disulfiram-ethanol-induced mortality but a single dose of ascorbic acid (2 mmole/kg) pretreatment had no effect. Increasing the ascorbic acid pretreatment dosage schedule to three days resulted in a reduction of disulfiram-ethanol mortality. These results indicate that thiamine HCl was more potent than ascorbic acid in reducing the mortality induced by acetaldehyde or disulfiram-ethanol. The effect of disulfiram on ascorbic acid or thiamine activity toward reduction of acetaldehyde-induced mortality was investigated. The same three-day pretreatment condition for disulfiram and ascorbic acid or thiamine was used in mice as in the above disulfiram-ethanol study. In the third day, acetaldehyde was given to the mice. Either vitamin was ineffective in reducing the mortality induced by disulfiram-acetaldehyde.